1. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, the present invention concerns a new class of insect inhibitory proteins comprising two different components, both of which are required for biological activity. The present invention concerns the construction of coleopteran-inhibitory crystal proteins, in particular CryET33/CryET34 and tIC100/tIC101 from Bacillus thuringiensis. Various methods of linking the proteins together, so that a single protein provides insect inhibitory activity, are disclosed. The use of nucleic acid sequences as diagnostic probes and templates for protein synthesis, and the use of polypeptides, fusion proteins, antibodies, and peptide fragments in various insect inhibitory, immunological, and diagnostic applications are also disclosed, as are methods of making and using nucleic acid sequences in the development of transgenic plant cells containing the nucleic acid sequences disclosed herein.
2. Description of the Related Art
Environmentally-sensitive methods for controlling or eradicating insect infestation are desirable in many instances, in particular when crops of commercial interest are at issue. The most widely used environmentally-sensitive insect inhibitory formulations developed in recent years have been composed of microbial pest control agents derived from the bacterium Bacillus thuringiensis. B. thuringiensis is well known in the art, and is characterized morphologically as a Gram-positive bacterium that produces crystal proteins or inclusion bodies which are aggregations of proteins specifically active against certain orders and species of insects. Many different strains of B. thuringiensis have been shown to produce insect inhibitory crystal proteins. Compositions including B. thuringiensis strains which produce insect inhibitory proteins have been commercially available and used as environmentally-acceptable pest control agents because they are quite toxic to the specific target insect, but are harmless to plants and other non-targeted organisms.
There are several B.t. crystal protein categories established based on primary structure information and the degree of protein similarities to one another. Over the past decade, research on the structure and function of B. thuringiensis crystal proteins has covered all of the major categories, and while these proteins differ in specific structure and function, general similarities in the structure and function are assumed. Based on the accumulated knowledge of B. thuringiensis insect inhibitory proteins, a generalized mode of action for B. thuringiensis insect inhibitory proteins has been created and includes: ingestion by the insect, solubilization in the insect midgut (a combination of stomach and small intestine), resistance to digestive enzymes sometimes with partial digestion actually “activating” the insect inhibitory protein, binding to the midgut cells, formation of a pore in the insect cells and the disruption of cellular homeostasis (English and Slatin, 1992).
Many of the δ-endotoxins are related to various degrees by similarities in their amino acid sequences. Historically, the proteins and the genes which encode them were classified based largely upon their spectrum of insect inhibitory activity. The review by Schnepf et al. (Microbiol. Mol. Biol. Rev. (1998) 62:775-806) discusses the genes and proteins that were identified in B. thuringiensis prior to 1998, and sets forth the most recent nomenclature and classification scheme as applied to B. thuringiensis insect inhibitory genes and proteins. Using older nomenclature classification schemes, cry1 genes were deemed to encode lepidopteran-inhibitory Cry1 proteins, cry2 genes were deemed to encode lepidopteran- and dipteran-inhibitory Cry2 proteins, cry3 genes were deemed to encode coleopteran-inhibitory Cry3 proteins, and cry4 genes were deemed to encode dipteran-inhibitory Cry4 proteins. However, new nomenclature systematically classifies the Cry proteins based upon amino acid sequence homology rather than upon insect target specificities. The classification scheme for many known proteins, not including allelic variations in individual proteins, including dendograms and full Bacillus thuringiensis protein lists is summarized and regularly updated at http://epunix.biols.susx.ac.uk/Home/Nil_Crickmore/Bt/index.html.
Most of the nearly 200 B.t. crystal proteins presently known have some degree of lepidopteran activity associated with them. The large majority of Bacillus thuringiensis insect inhibitory proteins which have been identified do not have coleopteran controlling activity. Therefore, it is particularly important, at least for commercial purposes, to identify additional coleopteran specific insect inhibitory proteins.
The B.t. proteins which have been identified as having coleopteran-inhibitory activity are either related to the Cry3 protein class, or are greater than about 74 kDa in size. (Berhnard, 1986; Donovan et al., 1988, 1992; Herrnstadt et al., 1986; Hofte et al., 1987, 1989; Kreig et al., 1983, 1984, 1987; McPherson et al., 1988; Sekar et al., 1987; Sick et al., 1990; U.S. Pat. No. 4,766,203; U.S. Pat. No. 4,771,131; U.S. Pat. No. 4,797,279; U.S. Pat. No. 4,910,016; U.S. Pat. No. 4,966,155; U.S. Pat. No. 4,966,765; U.S. Pat. No. 4,999,192; U.S. Pat. No. 5,006,336; U.S. Pat. No. 5,024,837; U.S. Pat. No. 5,055,293; U.S. Pat. No. 6,023,013; European Pat. Appl. Publ. No. 0318143; Eur. Pat. Appl. Publ. No. 0324254; Eur. Pat. Appl. Publ. No. 0382990; PCT Intl. Pat. Appl. Publ. No. WO 90/13651; Intl. Pat. Appl. Publ. No. WO 91/07481).
U.S. Pat. No. 6,063,756 disclosed Bacillus thuringiensis strains comprising novel crystal proteins which exhibit insect inhibitory activity against coleopteran insects including red flour beetle larvae (Tribolium castaneum) and Japanese beetle larvae (Popillia japonica). Also disclosed therein are novel B. thuringiensis genes, designated cryET33 and cryET34, which encode the coleopteran-inhibitory crystal proteins ET33 and ET34. cryET33 encodes the CryET33 (29-kDa) crystal protein, and the cryET34 gene encodes the 14-kDa CryET34 crystal protein. Also disclosed therein are methods of making and using transgenic cells comprising the novel nucleic acid sequences of the invention.
Rupar et al. (WO00/066742; PCT/US00/12136) describe still other expression systems isolated from Bacillus thuringiensis stains which express proteins, which, when present in approximately equimolar concentrations, exhibit Coleopteran insecticidal activity. In particular, a binary toxin system referred to as CryET80 and CryET76, ET76 being about 44 kDa and ET80 being about 14 kDa, are effective in controlling corn rootworms.
Narva et al. (U.S. patent application Ser. No. 09/378,088; WO01/14417(A2); PCT/US00/22942) disclose yet at least one other coleopteran inhibitory binary toxin exhibiting corn rootworm controlling bioactivity, isolated from Bacillus thuringiensis, and describe the construction of a fusion between the two components of the toxin, but failed do demonstrate any bioactivity of this fusion.
It would be useful to provide a protein to plants which exhibits coleopteran-inhibitory activity, which is less than about 74-kDa in size, which is expressed from a single open reading frame in order to, at least in plants, ensure simultaneous expression, and in particular in plants, in consideration of conservation of the genetic elements, create an easier means for breeding purposes.